1. Field of the Invention
The invention relates to the field of high sensitivity detection of molecules, especially biomolecules. The invention also relates to using fluorescent labels.
2. General Background and State of the Art
Ultrasensitive immunoassay methods are developed and used in clinical diagnostics to measure extremely low concentrations of specific compounds in highly complex samples. Although the sensitivity, reliability, rapidity, simplicity, and cost of these methods have steadily improved, further improvements are still needed and possible (Hampl J, et al., Upconverting phosphor reporters in chromatographic assays. Analytical Biochemistry, 2001; 288, 176-187; Unger M. et al, Single-molecule fluorescence observed with mercury lamp illumination. Biotechniques 1999; 27:1008-1014; and Weiss S. Fluorescence spectroscopy of single biomolecules. Science 1999; 283:1676-1683). The current trend toward miniaturized, multianalyte methods has introduced its own challenges and requirements for immunoassay technology (Taylor J R, et al., Probing specific sequences of single DNA molecules with bioconjugated fluorescent nanoparticles. Anal Chem 2000; 72:1979-1986; and Zijlmans, et al., Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology. Anal Biochem 1999; 267:30-36). The interest in new label technologies has especially increased because none of the commonly used direct or enzyme-amplified radioactive, colorimetric, luminescent, or fluorescent reporters fulfills all of the requirements for an ideal label, including specific activity, size, nontoxicity, cost, stability, localization, and detection. Directly detectable labels such as fluorophores suffer from limited sensitivity, and enzyme-amplified or dissociation-enhanced methods lose spatial information.
Recently, new detection methods based on high specific-activity particulate labels, such as quantum dots, luminescent inorganic crystals, up-converting phosphors, fluorescent nanoparticles, and plasmon resonant particles, have been introduced to respond to future demands for clinical diagnostics and biological, genomic, and pharmaceutical research. These submicrometer-sized labels are coupled to specific binding reagents such as nucleic acid probes, receptors, lectins, enzymes, and antibodies to detect specific molecules with sensitivities equal to or better than the best conventional labels available. In spite of the large molecular size and obvious stearic problems, these particular labels have also been used successfully in solid-phase immunoassays. It has been recognized, however, that the production, colloidal stability, and nonspecific binding of particle-protein bioconjugates may still require further improvements.
Time-resolved fluorometry and lanthanide labels were introduced for immunoassays 20 years ago. Since then, the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA®) technology has been known as one of the most sensitive and reliable immunoassay platforms. The research on intrinsically fluorescent, inert, and stable lanthanide chelate and cryptate labels has led to the development of novel homogeneous and heterogeneous assays that are expected to be introduced into routine clinical diagnostics. Moreover, an advanced dissociation-enhanced technology, based on lanthanide cofluorescence, which amplifies the long-lifetime fluorescence of europium(III), terbium(III), samarium(III), and dysprosium(III), has been known. A unique feature of lanthanide chelate fluorescence, the absence of self-quenching effects from multiple labeling, makes them ideal and suitable for high-density cluster labels such as dyed latex nanoparticles. The highly fluorescent chelates used in the DELFIA technology can also be used in fluorescent lanthanide(III) chelate nanoparticle because the hydrophobic environment inside the latex protects the fluorescent chelates from environmental effects, such as solvent quenching, and stabilizes the kinetically weak complexes. The adaptation of appropriate chelates for all four lanthanides would enable a nanoparticle-based, quadruple-labeling technology with an extremely low detection limit and a direct, surface readout measurement.
Detecting low levels of target marker in a sample using classic fluorochrome is sometimes difficult and prone to errors because specific fluorescence signals tend to be low and are usually mixed with nonspecific signals. Furthermore, autofluorescence produced from specimen can cause interference. The fluorescence half-life of complex chelate of lanthanide elements—e.g., europium (EU)—is as much as six orders of magitude longer than conventional fluorescent labels. Consequently, the emission from lanthanide chelate can be distinguished from background fluorescence (which has a short decay half-life) by using a time-resolved fluorometer with an appropriate delay, counting, and cycle times. This unique dye confers luminescence with a decay time of >500 μs, far longer than that of conventional fluorescent probes or autofluorescent samples, typically having decay times of <50 μs. Thus, time-resolved fluorometry can virtually eliminate autofluorescence.
These europium luminescent dyes feature long-wavelength emissions (˜610 nm) that are well separated from the excitation peak (˜365 nm). This unusually large Stokes shift permits the use of filter combinations that effectively isolate the desired luminescence signal (Harma et al., Europium nanoparticles and time-resolved fluorescence of ultrasensitive detection of prostate-specific antigen. 2001; 47:561-568). In the DELFIA system, lanthanide ions are dissociated from the chelating structure into the fluorescence enhancement solution. This additional enhancing step is required to provide an environment that effectively eliminates quenching water and contains energy-absorbing chelating compounds to transfer energy further to lanthanide ions. The lanthanide phosphors can be detected directly without any enhancing steps due to the water-protecting crystal structure. The disadvantage of using the lanthanide phosphors is the lack of light-absorbing groups that effectively transfer the absorbed energy to the lanthanide ions. Frank and Sundberg realized, in the late 1970s, that by combining these properties into a latex particle, fluorescent particles with a very high specific activity could be prepared. They prepared latex particles which contained a thenoyltrifluoroacetone lanthanide chelate complexed with tri-n-octylphosphine oxide (naphtoyltrifluoroacetone in the DELFIA technology complexed with tri-n-octylphosphine oxide) providing particles without quenching effects but having a light-absorbing group inside the particle. The polymer shell efficiently removes fluorescence-quenching water from the vicinity of the chelate by producing a hydrophobic environment. Extremely sensitive assays can be carried out using such particle labels (Härmä et al., 2001, Soukka et al., 2001a). These nanosized polymer labels contain 30,000-2,000,000 europium molecules entrapped by β-diketones, which have one of the highest quantum yields of the known lanthanide chelators (Harma et al., Europium nanoparticles and time-resolved fluorescence of ultrasensitive detection of prostate-specific antigen. 2001; 47:561-568). This encapsulation has no negative effect on the fluorescence efficiency. For a 100 nm size europium particle, the fluorescence yield is equivalent to about 3,000 molecules of fluorescein. Phycobiliprotein B-PE (perhaps the most fluorescent substance known) has a fluorescence yield equivalent to about 30 fluorescein molecules. Since a 100 nm particle is about 10 times the diameter of phycobiliprotein B-PE and a thousand times greater in volume/mass, these europium particles are 100 times more fluorescent than B-PE on a molar basis. This particle will give ultrasensitivity for assay because of its great fluorescence, broad stoke shift and long-lived luminescence. Encapsulation: 30,000-2,000,000 europium molecules in a single particle.